中国明对虾病原检测新方法及酚氧化酶通路相关基因的研究

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	中国明对虾病原检测新方法及酚氧化酶通路相关基因的研究
其他题名	Research on novel detection methods for pathogen and phenoloxidase pathway genes in Fenneropenaeus chinensis
	高宏伟
学位类型	博士
	2008-05-30
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	中国明对虾 Propo 丝氨酸蛋白酶fcsp3 基因克隆表达分析 Lamp 检测方法
摘要	对虾病害在世界范围内肆虐，给水产养殖和沿海农村经济造成了重大损失。在水产养殖的实践中快速检测水产动物的病害并及时采取隔离等措施对于控制病害尤为重要，其中关键的环节就是快速检测出病害，并在对虾免疫机制上寻找对虾疾病防治的有效方法。研究表明当对虾等甲壳动物受到外界病原刺激时，极微量的微生物多糖就可以激活proPO系统。激活过程中涉及和产生一系列活性物质，如黑色素、酚氧化酶原激活因子（PPA）、模式识别蛋白（BGBP、PGBP、LGBP、LBP）及其膜上受体和A2巨球蛋白等，它们可通过多种方式参与防御反应，包括提供调理素，促进血细胞吞噬作用，形成结节或包囊以及介导凝集和凝固，产生杀菌物质并且黑色素化。黑色素常常在节肢动物的体表形成黑色斑点，形成的色素沉着对机体起到保护作用。所以，酚氧化酶原激活的级联反应是节肢动物免疫的关键因素。本论文研究开发了以环等温介导技术（LAMP）为基础的检测对虾白斑病毒（WSSV）和鳗弧菌（V. anguillarum）的快速检测方法。并从对虾对病害的免疫机制为切入点，从中国明对虾体内克隆了酚氧化酶原（PrpPO）和丝氨酸蛋白酶FcSP3这两个免疫系统中重要的基因，分析了它们的分子结构特征，组织分布及应答鳗弧菌病原刺激的表达变化模式。建立的对虾常见病害对虾白班病毒（WSSV）和鳗弧菌（V. anguillarum）的LAMP检测方法，经过实验比对和Blast检索，发现本研究中使用的引物，比已经报导的LAMP方法或者PCR方法具有更宽的检测范围（更低的假阴性）。检测WSSV的LAMP方法使用病毒的VP28基因设计引物，而鳗弧菌的检测方法使用empA基因设计引物。在方法中，首次提出加入UNG酶和dUTP的措施来预防污染，在实际检测中非常有效。LAMP方法与PCR检测方法的灵敏性比较也进行了研究，二者灵敏性相当。依据中国明对虾血液cDNA文库提供的部分片段信息，结合SMART-RACE技术，克隆了酚氧化酶原（PrpPO）基因，通过序列比对分析发现，PrpPO基因cDNA全长为3040 bp，其中开放阅读框2061 bp，编码686个氨基酸，其中推测的信号肽为12个氨基酸。推测的序列与斑节对虾（P. monodon）同源性为93%,与短钩对虾（P. semisulcatus.）同源性为92%。real time RT-PCR实验结果表明， ProPO在血细胞中的相对表达量最高，肝胰脏中表达量最低。弧菌刺激实验中注射弧菌，刺激了血细胞和淋巴器官中的ProPO mRNA显著增加，说明在血细胞和淋巴器官中存在快速反应的ProPO通路。而ProPO mRNA量在淋巴器官中在时间上早于血液中升至最高，说明该动物在在病原刚开始入侵的时候先有淋巴器官发挥主要的免疫作用，随着时间推移血细胞便变成主要的免疫器官。根据中国明对虾肝胰脏cDNA文库提供EST信息，经过SMART-RACE克隆了一个丝氨酸蛋白酶FcSP3基因，通过序列比对分析发现，该丝氨酸蛋白酶基因cDNA全长为1622 bp，其中开放阅读框1431 bp，编码477个氨基酸，其中推测的信号肽为22个氨基酸。推测的序列与疟蚊的丝氨酸蛋白酶（A. gambiae）同源性为33%,与丽蝇蛹集金小蜂的酚氧化酶原激活因子（N. vitripennis）同源性为32%，与东北大黑鳃金龟的酚氧化酶原激活因子（H. diomphalia）同源性为34%。淋巴器官中PPAⅡ表达量约为血液中表达量的47560倍，肝胰脏中的FCSP3表达量为血细胞表达量的6226倍。鳗弧菌注射对虾后，淋巴器官中刺激组和对照组FcSP3的mRNA量在刺激后6小时显著降低，但是刺激组的表达量明显高于对照组。刺激组的血细胞与肝胰脏中FcSP3 mRNA的相对表达量增高。而病原刺激后的血液与肝胰脏中的FcSP3 mRNA的增长趋势也在时间上先与ProPO mRNA。这说明FcSP3对ProPO有正调控的作用，但这个调控有一个时间差，并且在不同组织中有不同的调控效率。
其他摘要	Shrimp cultivation has been suffering great losses due to the outbreak of diseases. To develop rapid detection of aquatic animal diseases and to take quatantine measures in time to control disease were equally important, in which the key step is to develop rapid detection method and to further reveal immune mechanism and find out effective method to control shrimp disease. Researches showed that when shrimp and other crustaceans were stimulated by outside pathogens, ProPO system in crustaceans would be activated a very small amount of microbial polysaccharides. A series of active substances, such as melanoma, prophenoloxidase activator factor (PPA), pattern recognition protein (BGBP, PGBP, LGBP, LBP) and membrane Giant globulin receptor and A2 were involved in the activation process. All these proteins participate in defense response, including the provision of conditioning, and promote blood cell phagocytosis, the formation of cysts and nodules or lectin-mediated and solidification, melanin production. Melanoma form black dot on the arthropod surface, which plays a role of body protector. Therefore, the reaction in prophenoloxidase path way is the key factor for the immune cascade arthropod. In the thesis, the loop-mediated isothermal amplification (LAMP) method for rapidly detecting white spot syndrome virus species and V. anguillarum in shrimp were found.And two key genes of phenoloxidase system (prophenoloxidase and Serine protease FCSP3 ) related to the immune system in Chinese shrimp F. chinensis have been clone and analyzed. Fragments of the VP28 gene of the WSSV virus and empA gene of V. anguillarum could be individually amplified at 65 ℃ in the presence of a primer mixture and Bst DNA polymerase. Amplification products were detected by visual inspection and agarose gel electrophoresis. The specificity of WSSV LAMP primers was demonstrated by the absence of amplification of DNA from healthy shrimp, and specificity of V. anguillarum LAMP primers was confirmed by ten non- V. anguillarum Bacteria. Compared with the performance of the established PCR and LAMP assay, both two developed LAMP methods had appeared to have wider spectrum for WSSV virus and V. anguillarum in detection. The prophenoloxidase (ProPO) gene was cloned from haemocytes of Chinese shrimp F. chinensis by Rapid Amplification Complementary DNA Ends (RACE) method. The full-length cDNA of ProPO consists of 3040 bp with a 2061 bp Open Reading Frame (ORF), which encodes 686 amino acids, and a predicted signal peptide of 12 amino acids. It showed 93% and 92% homology with those of P. monodon and P. semisulcatus. To understand its function, amounts of ProPO expressions in different tissues were studied by real-time PCR after challenged by V. anguillarum. The results showed that the expression level of ProPO in haemocytes was highest among three studied tissues which were haemocytes, hepatopancreas and lymphoid organ. After V. anguillarum injection, ProPO in haemocytes was observed at a lower mRNA level was observed at 6 hour post injection (hpi) compared with that of control, and then increased. The level graduately grow until it reached the peak at 12 hpi. In lymphoid organ of shrimp, the ProPO level was up-regulated to four times as normal level at 6 hpi, followed by a down-regulation. In all three organizations tissues, the controls group had changes less than the infected group, and the differences were significant. According to EST of hepatopancreas cDNA library of F. chinensis, a serine protease was cloned using RACE method. The full-length cDNA of Serine protease FCSP3 consists of 1622 bp with a 1431 bp ORF, which encodes 477 amino acids and a signal peptide of 22 amino acids. This gene show a 33% homology with Serine protease of A. gambiae, and 32% homology with prophenoloxidase activator factor of N. vitripennis, 34% homology with prophenoloxidase activator factor of H. diomphalia. The expression of serine protease was highest in the lymphoid organs, was lowest in haemocytes, The expression of FcSP3 in Lymphoid s after V. anguillarum injection increased significantly from 12 hpi to 72 hpi, while the control group changed a little. In lymphoid organ of shrimp, the FcSP3 level was down-regulated to 1/10000 times as normal level at 6 hpi, followed a sustaining low level . In hepatopancreas of shrimp, the FCSP3 level increased remarkable to 1.3 times as normal level, then droped back to normal at 24 hpi.
页数	118
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/781
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	高宏伟. 中国明对虾病原检测新方法及酚氧化酶通路相关基因的研究[D]. 海洋研究所. 中国科学院海洋研究所,2008.

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